The inadequate supply of human organs has created a strong interest in the use of non-primate organs for clinical transplantation. Unfortunately, vigorous immune reactions prevent their current use in humans. Genetic approaches have made great strides in overcoming some of the barriers arising from the presence of highly immunogenic xenoantigens and species incompatibilities in complement regulation. While these genetically engineered organs should elicit a reduced xenoreaction in humans, it is likely that residual xenoantigens will elicit significant immune responses that are distinct from those observed with human allografts. In particular, a vigorous T cell- independent antibody response is a unique and dominant feature in many models of xenograft rejection. We have turned to a rodent model of xenotransplantation, concordant hamster hearts-into-Lewis rats, to investigate the immunology of antibody-driven xenograft rejection. We have determined that the combination of two immunosuppressants, leflunomide and cyclosporine, completely inhibits immune xenoreactions in this model. We further observed that control of early acute xenograft rejection with this combination of immunosuppressants uncovers a late xenoreactivity that can be prevented solely by cyclosporine. Cessation of cyclosporine therapy results in a histologically novel, delayed xenograft rejection that is characterized, not by T cells, but by IgM deposition and an intense infiltrate of macrophages. The specific aims of this proposal center on, first, defining the role of T cells in delayed xenograft rejection; second, testing whether strategies that induce peripheral tolerance to allografts can induce long-term survival of xenografts in recipients pretreated with leflunomide plus cyclosporine; third, providing a cellular and molecular explanation for how pharmacological interdiction of early T-independent responses can result in a modified xenoreactivity that is T cell- dependent. Specifically, we will test whether the apparent loss of T cell-independent xenoreactivity is due to clonal anergy and/or deletion of a subset of B cells producing xenoreactive antibodies. Our proposed study is directly relevant to the goal of designing and transplanting transgenic xenografts that will survive and function with minimal immunosuppression in immunologically competent humans.